Triglyceride-inflammation score established on account of random survival forest for predicting survival in patients with nasopharyngeal carcinoma: a retrospective study

Objective This study aimed to establish an effective prognostic model based on triglyceride and inflammatory markers, including neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR), to predict overall survival (OS) in patients with nasopharyngeal carcinoma (NPC). Additionally, we aimed to explore the interaction and mediation between these biomarkers in their association with OS. Methods A retrospective review was conducted on 259 NPC patients who had blood lipid markers, including triglyceride and total cholesterol, as well as parameters of peripheral blood cells measured before treatment. These patients were followed up for over 5 years, and randomly divided into a training set (n=155) and a validation set (n=104). The triglyceride-inflammation (TI) score was developed using the random survival forest (RSF) algorithm. Subsequently, a nomogram was created. The performance of the prognostic model was measured by the concordance index (C-index), time-dependent receiver operating characteristic (ROC) curve, and decision curve analysis (DCA). The interaction and mediation between the biomarkers were further analyzed. Bioinformatics analysis based on the GEO dataset was used to investigate the association between triglyceride metabolism and immune cell infiltration. Results The C-index of the TI score was 0.806 in the training set, 0.759 in the validation set, and 0.808 in the entire set. The area under the curve of time-dependent ROC of TI score in predicting survival at 1, 3, and 5 years were 0.741, 0.847, and 0.871 respectively in the training set, and 0.811, 0.837, and 0.758 in the validation set, then 0.771, 0.848, and 0.862 in the entire set, suggesting that TI score had excellent performance in predicting OS in NPC patients. Patients with stage T1-T2 or M0 had significantly lower TI scores, NLR, and PLR, and higher LMR compared to those with stage T3-T3 or M1, respectively. The nomogram, which integrated age, sex, clinical stage, and TI score, demonstrated good clinical usefulness and predictive ability, as evaluated by the DCA. Significant interactions were found between triglyceride and NLR and platelet, but triglyceride did not exhibit any medicating effects in the inflammatory markers. Additionally, NPC tissues with active triglyceride synthesis exhibited high immune cell infiltration. Conclusion The TI score based on RSF represents a potential prognostic factor for NPC patients, offering convenience and economic advantages. The interaction between triglyceride and NLR may be attributed to the effect of triglyceride metabolism on immune response.


Introduction
Nasopharyngeal carcinoma (NPC) is a malignant head and neck tumor that has a high incidence in southern China and Southeast Asia (1).There is a double or triple incidence of NPC among males compared to females in most populations (1,2).According to a comprehensive assessment of the global cancer burden in 2022 (3) conducted by CANCER TODAY of the International Agency for Research on Cancer, a branch of the World Health Organization, the worldwide age-standardized rates of NPC were 1.9 per 100 000 person-years for males and 0.73 per 100 000 person-years for females.In Asia, the age-standardized rates of NPC were higher, with 2.6 per 100 000 person-years for males and 0.98 per 100 000 person-years for females.Radiation therapy is sensitive to NPC.Due to individual heterogeneity, patients with the same TNM stage may have different outcomes.The identification of serum tumor markers could aid in the noninvasive prediction of prognosis for NPC.
Reprogramming cellular metabolism is a key feature of the rapid proliferation of cancer cells (4), and the deregulation of lipid metabolism is one of the most common metabolic changes.We analyzed the lipid spectrum in the plasma of the subjects using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and found significant differences in the lipid profiles between NPC patients and healthy controls (5).Previous studies have reported that pretreatment serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) are negative and favorable independent prognostic factors for NPC, respectively (6,7).Chen et al. utilized widely targeted lipidomics to measure the plasma lipid profiles of patients with locoregionally advanced NPC.They identified six plasma lipid predictors for survival, which were significantly associated with inflammation-related markers such as C-reactive protein and white blood cell (WBC) count (8).
Tumor-promoting inflammation is also an enabling characteristic of cancer.Studies have reported that the inflammatory markers including neutrophil-to-lymphocyte ratio (NLR), lymphocyte-tomonocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) can reflect the body's inflammatory status and have prognostic value in various cancers (9)(10)(11)(12).In addition, it has been observed that a lower PLR is significantly associated with a higher pathological response rate for neoadjuvant combination therapy based on tislelizumab for muscle-invasive bladder cancer (13).Our previous research showed that high NLR, high PLR, and low LMR were significantly associated with poorer overall survival (OS) of NPC (14), similar to the studies conducted by Chen Y et al. (15) and Xu F et al. (16).
Patients with NPC should undergo routine blood tests to assess anemia, infection, and immune function, as well as blood lipid detection to address abnormal lipid levels promptly, mitigate cachexia, and prevent cardiovascular complications.It is easy to obtain through routine blood tests for lipid markers and the parameters of peripheral blood cell measurements in clinical practice.The integration of lipid markers and peripheral blood cell parameters allows for a comprehensive evaluation of prognostic value in NPC.However, most lipid or inflammatory markers were assessed separately, and the prognostic model was built using univariate and multivariate analyses, which had limitations in handling the collinearity among variables.The random survival forest (RSF) method, as the emerging machine learning algorithm, is not sensitive to multicollinearity and can significantly improve prediction performance.In this study, we aimed to construct a lipid-inflammation scoring model based on the RSF algorithm and estimate its performance in refining patient stratification, then compare it with the nutritional scores reported by others, such as controlling nutritional status (CONUT) score and prognostic nutritional index (PNI).Subsequently, a nomogram was constructed to predict the prognosis of NPC efficiently and conveniently.Additionally, the interaction and mediation between the biomarkers in their association with OS were analyzed.

Patients enrolled
The patient's data were collected retrospectively from February 2009 to December 2017 at Wuzhou Red Cross Hospital, China.The inclusion criteria for this study were as follows: (1) patients newly diagnosed with primary NPC based on pathological findings, (2) no prior anticancer treatment, and (3) fasting lipids test and routine blood test conducted before treatment.The exclusion criteria were: (1) prior malignancy, (2) metabolic diseases, and (3) infection or inflammation.The cancer TNM stage was determined according to the Chinese 2008 staging system (Supplementary Table 1).Patients with stages I-II received radiotherapy alone, while those with stages III-IV received radiotherapy in combination with chemotherapy.This study was approved by the Ethics Committee of Wuzhou Red Cross Hospital (NO.LL2021-53) and informed consent was waived due to its retrospective design.
The workflow chart of this study is shown in Figure 1.The NPC patients were randomly assigned to a training set and a validation set.The triglyceride-inflammation (TI) score based on the RSF algorithm was developed in the training set and validated in the validation set.The performance of the TI score was measured by the concordance index (C-index), time-dependent receiver operating characteristic (ROC) curve, and decision curve analysis (DCA).

Blood tests
Fasting blood samples were collected from each patient before initiating treatment.The blood lipid markers, including triglyceride (TG), total cholesterol (CHOL), high-density lipoproteincholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) and serum albumin were measured using a MODULAR DP automatic biochemical analyzer (Roche, Germany).The parameters of peripheral blood cells, including WBC, lymphocytes, neutrophils, monocytes, and platelets, were detected using an XE-2100 automatic hematology analyzer (Sysmex, Japan).All assays were performed according to the instruction manual of the reagent kits.

Follow-up
The OS was defined as the time from diagnosis to either death or the end of follow-up (December 31, 2021).

FIGURE 1
The workflow chart of this study.RCS, restricted cubic splines; RSF, random survival forest; TI, Triglyceride-inflammation.

Bioinformatics analysis of triglyceride metabolism
The GSE102349 dataset, downloaded from the Gene Expression Omnibus (GEO) website, is consists of 113 NPC tissues and prognostic data for 88 patients with progression-free survival (PFS) (17).The geneset for triglyceride metabolism included 2 key synthesis genes (AGPAT1 and DGAT1) and 3 key degradation genes (ATGL, HSL, and MGL).We performed a consensus clustering analysis on the geneset of triglyceride metabolism in the GSE102349 dataset and identified distinct patterns of triglyceride metabolism.We analyzed the differences in parameters of NPC patients with different triglyceride metabolism subtypes, including PFS, the immune microenvironment infiltration signature [based on EPIC (Estimate the Proportion of Immune and Cancer cells) (18) or MCPcounter (Microenvironment Cell Populations counter) (19)], and the immune score (calculated by ESTIMATE (20) or xCell (21) algorithms).

Statistical analysis
The statistical analyses and visualization of the data were performed using R version 4.3.0software from the R Foundation for Statistical Computing.To calculate the NLR, the neutrophil count was divided by the lymphocyte count.The LMR was calculated by dividing the lymphocyte count by the monocyte count.The PLR was calculated by dividing the platelet count by the lymphocyte count.The CONUT score (22) was calculated by the formula: serum albumin score + total lymphocyte count score + total cholesterol score (Supplementary Table 2).The PNI (23) was calculated by the formula: albumin (g/L) + 5 × lymphocyte count × 10 9 /L.The markers were presented as median (interquartile range), and differences between the two groups were compared using the Wilcoxon test.To assess the dose-response relationship between the markers and mortality in NPC patients, a restricted cubic splines (RCS) curve was applied using the R package "rms".Lipid-inflammation score was constructed using the RSF algorithm with the R package "randomForestSRC" (24).The prediction abilities of the lipidinflammation score were assessed using the C-index and the area under the curve (AUC) of the time-dependent ROC curve with the R package "timeROC" (25).The optimal cutoff values of the markers were determined using the R package "survminer".The OS of NPC patients between different groups was calculated using the Kaplan-Meier survival curve with the R packages "survminer" and "survival" (26).The hazard ratio (HR) and 95% confidence interval (CI) were estimated.A nomogram was constructed using the R package "rms", and its prognostic efficiency was evaluated using DCA with the R package "ggDCA".The interaction between inflammatory markers and triglyceride, as well as the mediation analysis, were performed using the R packages "visreg" (27) and "mediation" (28), respectively.The Consensus Clustering analysis was conducted using the R package "ConsensusClusterPlus" (29).The assessment of immune cell infiltration of NPC tissues was performed using the R package "IOBR" (30).A two-sided p-value < 0.05 was considered statistically significant.
These patients were randomly divided into a training set and a validation set at a ratio of 6:4 by using the sample function of the R package "base".There were no significant differences in the clinicopathologic parameters between the training set and validation set (Table 1).

High triglyceride, NLR, PLR, and low LMR are significantly associated with poor outcomes in NPC patients
To explore the association between circulating lipids, inflammatory markers, and the outcomes of NPC patients, we first utilized the RCS curve to model and visualize the relationship between lipids (triglyceride, cholesterol, HDL-C, and LDL-C), inflammatory markers (lymphocytes, neutrophils, monocytes, platelets, NLR, LMR, and PLR) with mortality in the entire set (Supplementary Figure 1).Our data demonstrated a gradual increase in the mortality risk of NPC patients with increasing levels of triglyceride, NLR, and PLR, while a gradual decrease in the mortality risk was observed with increasing lymphocyte count, platelet count, and LMR (p<0.05).There was no significant correlation between mortality and the markers of cholesterol, HDL-C, LDL-C, neutrophil count, and monocyte count (p>0.05).Additionally, Kaplan-Meier survival curve analysis revealed that NPC patients tend to have significantly poorer OS when exhibiting with higher levels of triglyceride, NLR, and PLR or lower levels of lymphocyte count and LMR (p<0.05)(Figure 2).

Triglyceride-inflammation score is a novel prognosticator for OS of NPC patients
Considering that the lymphocyte count is reflected by parameters NLR, LMR, and PLR, we utilized the RSF algorithm to construct the TI score in the training set, based on the level of triglyceride, NLR, LMR, and PLR which showed significant prognostic value for each NPC patient (Supplementary Figure 2).After parameter tuning, we found that when the ntree was set to 1000, the error rate of the model tended to be stable.The C-index was 0.806 (95%CI: 0.753-0.860) in the training set, 0.759 (95%CI:  3D-F).These results demonstrated that the TI score performed well in predicting survival with good sensitivity and specificity.In the entire set, the patients with stage T1-T2 or M0 exhibited significantly lower TI score, NLR, and PLR, as well as higher LMR compared to those with stage T3-T4 or M1, respectively (p < 0.05) (Table 2).Except for PLR, no differences were found in the levels of triglyceride, NLR, LMR, or TI score between male and female patients (Table 2).And stratified survival analysis by sex in the entire set (Supplementary Figure 3) showed that the C-index of TI score in the male and female subgroups were 0.780 (95%CI: 0.732-0.829)and 0.790 (95%CI: 0.697-0.882),respectively, without significant difference.These results indicated that the TI score might not be influenced by sex.
Among 259 NPC patients, 229 patients were detected for serum albumin before treatment.There were no significant differences in the clinicopathologic parameters between the albumin subset and the entire set (Supplementary Table 3).The survival analysis for the albumin subset showed that the C-index of CONUT score and PNI were 0.576 (95%CI: 0.513-0.640)and 0.591 (95%CI: 0.523-0.659),respectively; and the AUC in predicting survival at 1, 3, and 5 years were 0.693, 0.606 and 0.561 for CONUT score, and 0.717, 0.628 and 0.565 for PNI (Supplementary Figure 4).The prognostic efficacy of the CONUT score and PNI were worse than that of the TI score.

Nomogram based on TI score predicts the survival of NPC patients effectively
A COX regression model was further developed based on age, sex, overall stage, and TI score as variables.The resulting COX survival model was then used to create a nomogram, which was   displayed in Figure 4A.Calibration curves were generated to assess the accuracy of the model in predicting the 1-, 3-and 5-year OS and these curves demonstrated good discrimination (Figure 4B).To provide an unbiased estimate of model performance, we conducted an internal validation using a bootstrap resampling process with B=1000 (using the "validate" function of R package "rms").The Cindex and corrected C-index were calculated as 0.795 and 0.784, respectively.DCA was conducted to compare the predictive performance of the model incorporating age, sex, overall stage, and TI score (Figure 5).The net benefits of the prognostic model at 12, 36, and 60 months were found to be superior to those of the traditional model.

Statistical interaction but not mediation between triglyceride and NLR in the association with OS of NPC
Statistical interaction analyses of COX regression adjusted for age, sex, and overall stage revealed evidence of statistical interaction between triglyceride and NLR (HR=1.167,95% CI=1.062-1.283,p=0.001) (Figure 6A), as well as platelets (HR=0.998,95% CI=0.997-1.000,p=0.044) (Figure 6G).However, no significant interactions were observed between triglyceride and LMR, PLR, lymphocytes, neutrophils, and monocytes (Figures 6B-F; Supplementary Table 4).We established the regression models of survival outcome, observational variables (including age, sex, overall stage, and the parameters of peripheral blood cells), and mediator variable (triglyceride), and used the medicate function of R package "mediation" to estimate the average causal mediation effects (indirect effects) of NLR, LMR, PLR, lymphocytes, neutrophils, monocytes, or platelets on OS of NPC patients through the mediator triglyceride.The results indicated that triglyceride did not exhibit any medicating effects in the case of those inflammatory markers, respectively (Figure 7; Supplementary Table 5).

Computational analysis shows the association between intracellular triglyceride metabolism and immune cell infiltration
It is well known that the process of triglyceride metabolism is completed by a series of genes.To further explore the association between triglyceride metabolism and inflammatory markers (lymphocytes, neutrophils, and monocytes), computational analysis was conducted based on the GEO dataset GSE102349.We firstly performed RCS analysis and found a gradual increase in the disease progression risk of NPC patients with increasing expression of two key genes involved in the triglyceride anabolism (AGPAT1 and DGAT1), while a gradual decrease in the disease progression risk as observed with increasing expression of three key genes involved in the triglyceride catabolism (ATGL, HSL, and MGL) (Supplementary Figure 5).We then classified the NPC cases in GSE102349 into two subtypes according to the transcription level of the five triglyceride metabolism-related genes, via Consensus Clustering (Figures 8A, B).A statistical difference was found between the expressing patterns of the two subtypes.That is to say, NPC patients in subtype 1 showed a low triglyceride synthesis (lower expression of AGPAT1 and DGAT1) and high triglyceride degradation (higher expression of ATGL, HSL, and MGL) (p < 0.05), and reversely, NPC patients in subtype 2 showed a high triglyceride synthesis (higher expression of AGPAT1 and DGAT1) and low triglyceride degradation (lower expression of ATGL, HSL, and MGL) (p < 0.05) (Figure 8C).What's more, NPC patients in subtype 2 had poorer PFS compared to those in subtype 1 (Figure 8D), consistent with our hospital-based survival study in NPC patients with high serum levels of triglyceride (Figure 2A).Enlightened by the interaction between triglyceride and NLR, we next tried to explore the association between NPC subtypes and immune cell infiltration.It was noted that NPC subtype 1 exhibited higher proportions of B cells and T cells, as well as a higher immune score (Figures 9A-D), and the proportions of monocytic lineage and neutrophils were significantly higher in NPC subtype 1 than in subtype 2 (Figure 9B), indicating a significant association between intracellular triglyceride metabolism and immune cell infiltration in NPC tissues.However, further

Discussion
Nowadays, interaction among metabolism, inflammation, and cancer microenvironment, is becoming one of the hot spots in cancer research.In the present study, we first examined the correlation between blood triglyceride, inflammatory markers, and the OS of NPC patients, and found that high levels of triglyceride, NLR, and PLR, as well as low LMR, predicted poor survival outcomes of NPC patients.A machine learning model, namely TI score, was then developed by integrating the circulating level of triglyceride, NLR, LMR, and PLR, and showed favorable prognostic function in NPC, superior to the other score systems such as CONUT score or PNI.Statistical interaction was found between triglyceride and NLR in the association with OS of NPC patients.Additionally, computational analysis showed that NPC patients with low triglyceride synthesis and high triglyceride degradation were associated with higher infiltration Assessment of interaction between triglyceride and NLR (A), LMR (B), PLR (C), lymphocytes (D), neutrophils (E), monocytes (F), and platelets (G).NLR, neutrophil to lymphocyte ratio; LMR, lymphocyte to monocyte ratio; PLR, platelet to lymphocyte ratio. of B cells and T cells, and higher immune scores, proposing a potential interaction between triglyceride metabolism and NPC tumor microenvironment.
Metabolism dysregulation is a critical hallmark of cancer.Obesity and excessive consumption of high-fat diets can alter metabolism and cellular status in healthy tissues, increasing the susceptibility to cancer (31).What's more, tumor cells remodulate lipid metabolism in order to grow quickly and progress aggressively (32).Triglyceride, as the main constituents of human body fat, enhances tumor cell proliferation and growth in the context of hypoxia and hypoperfusion (33).Higher level of serum triglycerides is associated with increased risks of breast, lung, rectal, and thyroid cancer (34,35).Overactivation of triglyceride synthesis and downregulation of triglyceride degradation have been noted in the tumorigenesis of colorectal cancer (CRC) (36).Furthermore, high level of serum triglyceride is closely associated with poor prognosis in non-small cell lung cancer (37).Similar results are found in NPC.For instance, an Italian study reported that a high dietary intake of cholesterol could increase the risk of NPC (38).In our previous study, an epidemiological survey indicated that a higher intake of animal-based foods was associated with an elevated risk of NPC (39), and a hospital-based case-control study showed significantly different plasma lipidomic profiles between NPC patients and controls (5).Recent research also found that triglyceride was a potential risk factor for poor OS of non-metastatic NPC and eye metastases in male NPC patients (40,41).In line with the above studies, the present study showed that high serum triglyceride was positively associated with the poor survival of NPC patients, which might be a prognostic predictor of NPC.Considering there is no available public dataset containing OS information on NPC cases, we tried to analyze the association between triglyceride metabolism and progression-free survival in NPC based on the GEO dataset GSE102349.As shown by bioinformatics analysis, high intracellular triglyceride synthesis (higher expression of AGPAT1 and DGAT1) and low triglyceride degradation (lower expression of ATGL, HSL, and MGL) were positively related to the disease progression of NPC.This was consistent with our previous study, in which obvious lipid droplet accumulation and its abilities to promote cell proliferation and invasion were found in NPC (42), and siRNA silencing of ATGL exhibited an increase of lipid droplets in NPC cells (43).We attempted to feed the C17 NPC patient-derived xenograft (PDX) model with a high-fat diet for 14 days and found that the high-fat diet accelerated NPC proliferation (data unpublished).To some extent, it was indicated that peripheral blood triglyceride levels may affect the progression of NPC.
Blood inflammatory markers have a strong prognostic value in many types of cancer.Given their easy accessibility and low costs in clinical practice, biomarkers such as NLR, RLR, and LMR, are widely evaluated for their prognostic function.It has been shown that the NLR and PLR can reliably predict the OS and PFS in patients with ovarian, prostate, or head and neck cancers (9,11,12).A low LMR is also independently associated with worse OS in CRC patients (44).There are also accumulated reports about their application in NPC.NPC patients with higher NLR, PLR, systemic immune index, and systemic inflammation response index, and lower LMR experienced poorer OS or PFS (14)(15)(16)45).
In this study, single-marker analysis revealed that NLR and PLR were associated with an increased risk of death, while LMR was associated with a decreased risk of death in NPC, further supporting the roles of NLR, PLR, and LMR in the prognosis of NPC.Machine learning is widely applied in improving the accuracy of cancer susceptibility, recurrence, and survival prediction (46)(47)(48).As one of the popular machine learning algorithms, the tree-based RSF possessed a certain anti-noise ability due to the introduction of randomness.RSF models have been constructed for prognostic prediction of NPC, by applying microRNA, or tumor infiltrating lymphocytes (49,50).Yet, to our knowledge, there is no RSF model based on lipid metabolism and inflammation.In this study, we generated a TI score by RSF, based on the circulating triglyceride, NLR, PLR, and LMR, which were identified through univariate analysis.The TI score worked excellently not only in survival prediction but also showed a significant positive association with tumor burden and distant metastasis in NPC, indicating its potential power in clinical application.
It is well-known that remodification of metabolism, inflammation, and immune microenvironment are critical biomarkers of cancer (4), and there is a complicated crosstalk among the three.Serum triglyceride levels have been associated with oxidative stress and chronic inflammation, which may play a potential role in the proliferation and progression of cancer cells (51).Additionally, cholesterol and fatty acids can regulate T cell differentiation through the activation of multiple signals (52).Low-grade inflammation induced by obesity, hyperglycemia, and excessive lipid accumulation is usually systemic and can promote the risk of many different cancers (53,54).Interestingly, lipid-accumulating lung mesenchymal cells promote the metastasis of breast cancer cells by metabolic rewiring of cancer cells and natural killer cells, partially via the IL-1b-HILPDA-ATGL axis (55).Herein, our study indicated that there was an interaction between triglyceride and NLR, and NPC tissues with active triglyceride synthesis exhibited low immune cell infiltration, providing new clues for the mechanism of immune evasion in NPC.
Increasing evidence declare that lipids are associated with many immune cells and their phenotypic transformation, and tumor lipid metabolism is reprogrammed to promote immunosuppression, leading to immune escape (56).As the cancer-stromal interactions are intensified in the process of tumor progression, fatty acids secreted into the microenvironment would affect the function and phenotype of infiltrating immune cells (57).Studies showed that triglyceride-induced monocyte death by activating caspase-8/caspase-2/DNA damage pathways (58), and triglyceride-rich lipoproteininduced monocyte-derived dendritic cells activation through ApoB48R upregulation in a fatty acid-dependent manner (59).Moreover, the environment enriched in lipid-dependent fatty acids might induce the phenotype of tumor-associated macrophages (TAMs), which have the characteristics of M2-like macrophages and exert multiple pro-tumoral properties (60).Abnormal accumulation of cholesterol in TAMs induces upregulation of ABCA1/G1 cholesterol efflux receptors and induces mitochondrial damage through oxidation product 7-ketocholesterol, which leads to TAMs phagocytic fragility and inhibits tumor necrosis factor signaling in TAMs (61).Lipid molecules such as triglyceride and cholesterol not only play a role in the metabolism and signal transduction of cancer cells but also participate in the response of immune cells recruited by tumors.Therefore, further elucidating the complex interactions between the different cell types and how lipids change their responses to each other will open up a road for NPC patients to improve their treatment and outcome.
There are some limits in this study.First, some patients were not included in this study since the blood lipid test before treatment was not necessary.However, the age and sex distribution of the enrolled patients did not deviate from the characteristics of the population in high-risk areas, so there might be only limited selective bias in this cohort.Second, the generalizability of the TI score was somewhat restricted, since the data were retrospectively collected from a single hospital and the model has not undergone external validation.It will be essential to carry out a large-scale multicenter study for validation in the future.In the end, we developed a TI score via RSF, which was based on the convenience and cost-effectiveness of clinical parameters (triglyceride, NLR, RLR, and LMR), and generated a nomogram to accurately predict survival in NPC patients.What's more, we will further investigate the influence of triglyceride metabolism on NPC development, partially due to the dysregulation of immune cell infiltration, which might thread light on the personalized prognosis and treatment strategies of NPC.

3
FIGURE 3 Kaplan-Meier and time-dependent ROC curves analyses based on TI score of overall survival of NPC patients in the training set (A, D), validation set (B, E), and entire set (C, F).TI, Triglyceride-inflammation.

FIGURE 5
FIGURE 5 Comparison of decision curve analysis between the model based on age, sex, and overall stage and TI score-based model in 12, 36, and 60 months.TI, Triglyceride-inflammation.

8 Consensus
FIGURE 8 Consensus Clustering of triglyceride metabolism in GSE102349.(A) Consensus clustering matrix for k = 2. (B) Consensus clustering cumulative distribution function for k = 2-6.(C) Expression differences in the triglyceride metabolism genes between two subtypes.(D) Kaplan-Meier curves of progression-free survival for two subtypes in NPC.Subtype 1: low anabolism and high catabolism of triglyceride; Subtype 2: high anabolism and low catabolism of triglyceride.*p < 0.05, ***p < 0.001.

9
FIGURE 9 Immune status in two subtypes of triglyceride metabolism in the GSE102349 dataset of NPC.Differences in immune cell infiltration between the two subtypes were analyzed by using EPIC (A) or MCP-counter (B) signature.Differences in immune scores between the two subtypes were analyzed by using the ESTIMATE (C) and xCell (D) algorithms.Subtype 1: low anabolism and high catabolism of triglyceride; Subtype 2: high anabolism and low catabolism of triglyceride.TG: triglyceride, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no statistical significance.

TABLE 2
Association of pretreatment NLR, LMR, PLR, and TI score with clinicopathologic characteristics in the entire set of NPC patients [median (interquartile range)].